Intravital Microscopy for Hematopoietic Studies.

The bone marrow (BM) is home to numerous cell types arising from hematopoietic stem cells (HSCs) and nonhematopoietic mesenchymal stem cells, as well as stromal cell components. Together they form the BM microenvironment or HSC niche. HSCs critically depend on signaling from these niches to function and survive in the long term. Significant advances in imaging technologies over the past decade have permitted the study of the BM microenvironment in mice, particularly with the development of intravital microscopy (IVM), which provides a powerful method to study these cells in vivo and in real time. Still, there is a lot to be learnt about the interactions of individual HSCs with their environment - at steady state and under various stresses - and whether specific niches exist for distinct developing hematopoietic lineages. Here, we describe our protocol and techniques used to visualize transplanted HSCs in the mouse calvarium, using combined confocal and two-photon IVM.

Multiphoton intravital microscopy of rodents.

Tissues are heterogeneous with respect to cellular and non-cellular components and in the dynamic interactions between these elements. To study the behaviour and fate of individual cells in these complex tissues, intravital microscopy (IVM) techniques such as multiphoton microscopy have been developed to visualize intact and live tissues at cellular and subcellular resolution. IVM experiments have revealed unique insights into the dynamic interplay between different cell types and their local environment, and how this drives morphogenesis and homeostasis of tissues, inflammation and immune responses, and the development of various diseases. This Primer introduces researchers to IVM technologies, with a focus on multiphoton microscopy of rodents, and discusses challenges, solutions and practical tips on how to perform IVM. To illustrate the unique potential of IVM, several examples of results are highlighted. Finally, we discuss data reproducibility and how to handle big imaging data sets.

Author Correction: Dynamic responses of the haematopoietic stem cell niche to diverse stresses.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Enhanced human hematopoietic stem and progenitor cell engraftment by blocking donor T cell-mediated TNFα signaling.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative therapy, but the large number of HSCs required limits its widespread use. Host conditioning and donor cell composition are known to affect HSCT outcomes. However, the specific role that the posttransplantation signaling environment plays in donor HSC fate is poorly understood. To mimic clinical HSCT, we injected human umbilical cord blood (UCB) cells at different doses and compositions into immunodeficient NOD/SCID/IL-2Rgc-null (NSG) mice. Surprisingly, higher UCB cell doses inversely correlated with stem and progenitor cell engraftment. This observation was attributable to increased donor cell-derived inflammatory signals. Donor T cell-derived tumor necrosis factor-α (TNFα) was specifically found to directly impair the survival and division of transplanted HSCs and progenitor cells. Neutralizing donor T cell-derived TNFα in vivo increased short-term stem and progenitor cell engraftment, accelerated hematopoietic recovery, and altered donor immune cell compositions. This direct effect of TNFα on transplanted cells could be decoupled from the indirect effect of alleviating graft-versus-host disease (GVHD) by interleukin-6 (IL-6) blockade. Our study demonstrates that donor immune cell-derived inflammatory signals directly influence HSC fate, and provides new clinically relevant strategies to improve engraftment efficiency during HSCT.

Systematic tracking of altered haematopoiesis during sporozoite-mediated malaria development reveals multiple response points.

Haematopoiesis is the complex developmental process that maintains the turnover of all blood cell lineages. It critically depends on the correct functioning of rare, quiescent haematopoietic stem cells (HSCs) and more numerous, HSC-derived, highly proliferative and differentiating haematopoietic progenitor cells (HPCs). Infection is known to affect HSCs, with severe and chronic inflammatory stimuli leading to stem cell pool depletion, while acute, non-lethal infections exert transient and even potentiating effects. Both whether this paradigm applies to all infections and whether the HSC response is the dominant driver of the changes observed during stressed haematopoiesis remain open questions. We use a mouse model of malaria, based on natural, sporozoite-driven Plasmodium berghei infection, as an experimental platform to gain a global view of haematopoietic perturbations during infection progression. We observe coordinated responses by the most primitive HSCs and multiple HPCs, some starting before blood parasitaemia is detected. We show that, despite highly variable inter-host responses, primitive HSCs become highly proliferative, but mathematical modelling suggests that this alone is not sufficient to significantly impact the whole haematopoietic cascade. We observe that the dramatic expansion of Sca-1(+) progenitors results from combined proliferation of direct HSC progeny and phenotypic changes in downstream populations. We observe that the simultaneous perturbation of HSC/HPC population dynamics is coupled with early signs of anaemia onset. Our data uncover a complex relationship between Plasmodium and its host's haematopoiesis and raise the question whether the variable responses observed may affect the outcome of the infection itself and its long-term consequences on the host.

Tracking single cells in live animals using a photoconvertible near-infrared cell membrane label.

We describe a novel photoconversion technique to track individual cells in vivo using a commercial lipophilic membrane dye, DiR. We show that DiR exhibits a permanent fluorescence emission shift (photoconversion) after light exposure and does not reacquire the original color over time. Ratiometric imaging can be used to distinguish photoconverted from non-converted cells with high sensitivity. Combining the use of this photoconvertible dye with intravital microscopy, we tracked the division of individual hematopoietic stem/progenitor cells within the calvarium bone marrow of live mice. We also studied the peripheral differentiation of individual T cells by tracking the gain or loss of FoxP3-GFP expression, a marker of the immune suppressive function of CD4(+) T cells. With the near-infrared photoconvertible membrane dye, the entire visible spectral range is available for simultaneous use with other fluorescent proteins to monitor gene expression or to trace cell lineage commitment in vivo with high spatial and temporal resolution.

Adult stem cels and their niches.

Stem cells participate in dynamic physiologic systems that dictate the outcome of developmental events and organismal stress, Since these cells are fundamental to tissue maintenance and repair, the signals they receive play a critical role in the integrity of the organism. Much work has focused on stem cell identification and the molecular pathways involved in their regulation. Yet, we understand little about how these pathways achieve physiologically responsive stem cell functions. This chapter will review the state of our understanding of stem cells in the context of their microenvironment regarding the relation between stem cell niche dysfunction, carcinogenesis and aging.